저자는이 비디오를 제작 미주리 학술 지원 센터의 대학에서 크리에이티브 서비스 팀에게 감사하고 싶습니다. 이 작품은 국립 보건원의 아동 건강 및 인간 발달의 유니스 케네디 Shriver 국립 연구소, 교부금 번호 HD055231에 의해 지원되었다

1. 마우스 태반의 해부 <ol><li> 마우스의 결합 쌍을 설정합니다. </li><li> 다음과 같은 일의 각에서 copulatory 플러그의 존재를 확인합니다. 플러그가 감지되는 날은 0.5 일 이후의 성교 (dpc) 7 간주됩니다. </li><li> 태반은 D 10.5에 성숙 태반의 발달과 태반 가능한 시작의 깨끗한 절개로, 임신의 원하는 단계에서 수집 될 수 있습니다. 여기, 우리는 14.5 dpc을 보여줍니다. 주어진 볼륨에서 10.5 dpc에 40-60 태반, 그리고 14.5 dpc에 10-20 태반은 명확하게 볼 수 trophoblast 세포 레이어를 얻을 수 있습니다. </li><li> 임신 마우스를 안락사시켜야하고, 자궁 경부에서 절단, 자궁을 제거하고 mesometrium 따라. </li><li> 각 conceptus (그림 1A) 사이에 컷 날카로운 가위를 사용합니다. </li><li> 포셉의 두 쌍을 사용하여 절단 가장자리에 자궁 벽을 파악하고, 분열, myometrium (그림 1B)를 제거<html> . 한 쌍의 톱니 모양 (곡선 또는 직선) 적당히 고급 집게와 한 쌍의 아주 좋은 포셉 (# 5, 예를 들어)을 권장합니다. </li><li> 태반 맞은 편에 태반 노출, 집게 가위로 decidua를 엽니 다. 한 포셉 쌍과 자궁 조직을 파악하는 것은 분리 할 수있는 태반 (그림 1C) 아래에 다른 집게를 끼 웁니다. 만약 태반에서 필요한, ​​별도의 태아와 태아 막의. 또한 태반 조직을 잃지 않고 절개로 모든 decidual 조직을 제거 할 수는 없지만 추가 decidual 조직은 (창백한) 부드럽게 집게와 필링하여 태반의 표면에서 제거 할 수 있습니다. 우리는 더 정화를위한 Percoll 기울기에 의존합니다. </li><li> 모든 dissections이 완료 될 때까지 50 ML 원뿔 튜브 25 ML의 얼음 차가운 해리 버퍼에있는 각 해부 태반를 놓습니다. 나중에 단계에서, coarsely 해리 버퍼에 전송하기 전에 보 또는 면도날과 태반 세포를 이기다. </li></ol>trophoblast 세포의 tle &quot;&gt; 2. 절연 <ol><li> 45-60분을위한 37 ° C의 물에 대충 모자 원뿔 튜브 및 장소. 대략 10 분 pipetting하여 힘차게 분리 솔루션을 섞는다. 총 소화 시간이 면밀히 모니터해야하며 가능성이 몇 가지 시행 착오를 최적의 세포 복구를 달성하기 위해 필요합니다. 조직은 10 ML의 pipet 통과 할 수 있어야하지만, 조직의 표시 부분은 유지됩니다. Overdigestion 가난 세포 생존과 도금 효율이 높아집니다. 몇 세포의 clumps이 존재하는 경우 최적의 세포 생존과 도금 효율은 비교적 분리가 달성 될 때보다 발생합니다. 반대로, underdigestion는 percoll 분리에 따라 감소 순도로 이어질 것입니다. 적절한 소화 지점에 도달했을 때, 얼음에 장소 튜브 collagenase의 활동을 중지 할 수 있습니다. </li><li> 소화되지 않은 물질을 제거하고 5 분 @ 500 XG를 원심 분리기하는 셀 스트레이너를 통해 솔루션을 전달합니다. </li><li> 씻어 제거10 ML 세척 솔루션의 표면에 뜨는 및 resuspend 세포. 5 분 @ 500 XG를 원심 분리기. </li><li> 원심 분리하는 동안, 9.6 ML percoll, 13.4 ML 세척 솔루션과 고속 원심 분리에 적합한 50 ML 튜브에 1.1 ML 10 배 중간 199 혼합하여 percoll 솔루션을 준비합니다. </li><li> 2 ML 세척 용액에 표면에 뜨는 및 resuspend 세포 펠렛을 제거합니다. </li><li> 솔루션을 percoll와 4에서 30 분 @ 30,000 XG를 원심 분리기에 셀을 추가 ° C. </li><li> 기울기를 방해하지 않으면 서 원심 분리기에서 튜브를 제거합니다. 밝은 광원 앞의 그라데이션, 장소 튜브를 시각화합니다. trophoblast 밴드 (그림 2)을 찾아 아래 적혈구 밴드를 방해하지 돌보는, pipet을 대기음. trophoblast 대역의 볼륨은 5-8 ML 될 것입니다. </li><li> 50 ML 원뿔 튜브에 trophoblast 세포를 전송합니다. 45ml의 총 볼륨에 대한 솔루션을 씻어 추가 할 수 있습니다. </li><li> 5 분 @ 500 XG를 원심 분리기. 표면에 뜨는 제거하고, 문화 메디에 resuspend음 ... (NCTC-135). 세포는 37 ° C 5 % CO 2에서 얇은 층 matrigel 또는 조직 배양 플라스틱에 배양 할 수 있습니다. 일 10.5에서 각 태반은 80-90% 긍정적 인 cytokeratin되는 대략 50,000 라이브 세포를 산출. </li></ol> 3. Trophoblast의 침공 분석 <ol><li> 37 각 matrigel 챔버 및 품다 1-2 ML 문화 매체 (NCTC-135)을 추가 °, 30-90 분에 대한 5% CO 2. </li><li> 미디어를 제거하고, 상부 챔버에 2ml 매체에 1.5 ML × 10 5 갓 고립 된 세포를 추가 할 수 있습니다. 낮은 막 표면을 충당하기 위해 하부 챔버에 1 ML 매체를 추가 할 수 있습니다. </li><li> 18~24시간에 대한 삽입의 문화 세포. </li><li> 거품 면봉을 사용하여 삽입에서 다쳤고, 셀 및 matrigel. </li><li> 2 ML PBS의 상단과 챔버의 바닥을 씻어. </li><li> PBS 1 ML 4 % paraformaldehyde에 5 분 수정. </li><li> 400 μl DAPI (300nM)와 하부 챔버 채워 청바지 30 분에 품다. </li><li> PBS에서 간단히 세 번 씻으십시오. </li><li&gt;을 날카로운 메스 블레이드와 삽입에서 세포막을 제거합니다. </li><li> 슬라이드 마운트 매체 (PBS의 50 % 글리세롤)의 10 μl 방울을 추가합니다. 집게를 사용하여 드롭에 막 놓여 있었다. 매체의 또 다른 10 μl 방울을 추가 한 다음 조심스럽게 coverslip. </li><li> 사진 및 count 세포. 사용중인 현미경에 대한 적절한 방향 막 (아래 셀 사이드 업 또는 셀 측)에 있는지 확인하십시오. 이미지 J 소프트웨어 (보건 국립 연구소)이 목적을 위해 사용할 수있는 수동 계산 기능을 포함 할 수 있습니다 어도비 포토샵 또는 수동 카운터 있습니다. </li></ol> 4. 대표 결과 <img src="/files/ftp_upload/3202/3202fig1.jpg" alt="그림 1" /> 그림 1. 마우스 태반의 해부. (A) 자궁이 mesometrial 표면을 따라, (노란색) oviducts 따라 절단하여 제거하고 자궁 경부 (아래)에 있습니다. 개인 conceptuses 그런 다음 그림과 같이 가위로 절단하여 분리 할 수​​ 있습니다. (B) 자궁<html> 벽은 컷 자궁 표면에있는 두 집게을 배치하고, 태아 근처에 떨어진 태반에서 반 mesometrial 쪽을 따라 찢어 의해 제거됩니다. (C) 태반은 다음 집게로 물체를 움켜 태반과 자궁 사이에 서로를 슬라이딩으로 절연되어 있습니다. 탯줄 및 기타 extraembryonic 멤브레인은 다음 태반에서 분리 할 수​​ 있습니다. <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="그림 2" /> trophoblast 세포의 그림 2. Percoll 분리. 원심 분리 후, 세 가지 주요 밴드가 왼쪽에 만화 같이 볼 수 있으며, 사진 오른쪽 것입니다. 위 밴드는 세포 파편과 섬유 아세포를 포함하고 가장 명백 할 것이다. 적혈구가 포함 된 하단에 꼭 (빨간색) 밴드는 확산 trophoblast 밴드 아래에 표시됩니다. 단지 사진의 화살표의 왼쪽에 표시된 두에서와 같이 셀을 합산 가능성이, trophoblast 밴드에서 볼 수 있습니다. ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="그림 3" /> 그림 3. 침윤성 trophoblast 세포 (흰색 화살표)는 멤브레인의 바닥 표면에 게재 matrigel 층과 막 숨구멍을 통과하게된다. 모공은 (검은 색 화살촉)도 볼 수 있습니다. 전체 막 표면 또는 대표 샘플 지역에 세포의 수는 계산 될 수 있습니다. 형광 DAPI 핵 염색의 그레이 스케일 이미지는 세포의 시각화를 개선하기 위해 반전되었습니다. <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="그림 4" /> 그림 4. Trophoblast 세포는 식별 및 격리 인구의 순도는 cytokeratin 7 (센터, 마젠타)에 immunostaining에 의해 평가 될 수있다. 세포는 DAPI (위, 청색)로 counterstained 있습니다. 통합 이미지 하단에.

<ol>
	<li>Thordarson, G., Folger, P., Talamantes, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+placental+cell+culture+system+for+studying+the+control+of+mouse+placental+lactogen+II+secretion.">Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion.</a> Placenta. 8, 573-585 (1987).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods. Mol. Med. 121, 203-217 (2006).</li><li>Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+in+vitro+differentiation+of+cytotrophoblasts+from+human+term+placentae.">Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.</a> Endocrinology. 118, 1567-1582 (1986).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Librach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=92-kD+type+IV+collagenase+mediates+invasion+of+human+cytotrophoblasts.">92-kD type IV collagenase mediates invasion of human cytotrophoblasts.</a> J. Cell. Biol. 113, 437-449 (1991).</li><li>Schulz, L. C., Widmaier, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leptin+on+mouse+trophoblast+cell+invasion.">The effect of leptin on mouse trophoblast cell invasion.</a> Biol. Reprod. 71, 1963-1967 (2004).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

우리는 공개 할 아무 것도 없습니다.

이 동영상에서는, 우리는 마우스 태반에서 trophoblast 세포의 절연을 보여줍니다. 우리는 trophoblast의 침략에의 시험 관내 분석을 보여줍니다. 이 절차를 사용은 크게,하지만 완전히 trophoblast 세포의 순수한 인구는 얻을 수 있습니다. 더 순도가 필요한 경우 기본 인간 trophoblast 세포에 대한 설명 되었기 때문에, 자기 비드 분리 또는 유동 세포 계측법은 수행 할 수 있습니다. collagenase 분리 단계의 적절?...

Specific reagents and equipment:
Wash Solution (filter sterilize after mixing)
<table border='1'>
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>50ml</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>2.383 g</td>
 <td>0.02M</td>
 </tr>
 <tr>
 <td>sodium bicarbonate</td>
 <td>0.42 g</td>
 <td>0.01M</td>
 </tr>
 <tr>
 <td>Penicllin-Streptomycin</td>
 <td>5 mL</td>
 <td>100 U- 100 &mu;g /ml</td>
 </tr>
 <tr>
 <td>Sterile dH20</td>
 <td>to 500 mL</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

Dissociation solution (make fresh and filter sterilize)
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>Wash Solution</td>
 <td>100 mL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>1 vial (2000 U)</td>
 <td>20 U/mL</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>100 mg</td>
 <td>125 digestion units/mL</td>
 </tr>
 </tbody>
</table>


<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma</td>
 <td>C9891</td>
 <td>This is Clostridium collagenase.
Bovine pancreatic collagenase would likely work as well</td>
 </tr>
 <tr>
 <td>NCTC-135</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>Add FBS to 10%</td>
 </tr>
 <tr>
 <td>Matrigel invasion chambers</td>
 <td>BD Biosciences</td>
 <td>354481</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>Sigma</td>
 <td>M9163</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m Cell strainer</td>
 <td>Fisher</td>
 <td>22363549</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibody to cytokeratin 7</td>
 <td>DAKO</td>
 <td>M7018</td>
 <td>Used at 1:100 dilution</td>
 </tr>
 </tbody>
</table>

Equipment Standard cell culture equipment including a CO2 incubator is required. In addition, a centrifuge rotor capable of spinning 50 ml tubes at 30,000 x g is needed. Note that this exceeds the maximum speed of most conical centrifuge tubes and will likely require an Oak Ridge style tube.

isolation of primary mouse trophoblast cells and trophoblast invasion assay

태반은 태아에게 영양분, 가스 및 성장 요인의 운송뿐만 아니라 폐기물의 제거를 담당하고 있습니다. 따라서, 태반 개발에 결함이 태아와 어머니에 대한 중요한 결과를 가지고 있고, 배아 lethality의 주요 원인입니다. 태반의 태아 부분의 주요 세포 유형은 trophoblast입니다. 기본 마우스 태반 trophoblast 세포는 정상과 비정상 태반 개발을 공부에 유용한 도구이며, 세포 라인과는 달리, 격리 및 임신의 특정 단계에서 trophoblast을 공부하는 데 사용할 수 있습니다. 또한, 유전자 변형 생쥐에서 trophoblast의 주 문화는 태반 세포에서 특정 유전자의 역할을 연구하는 데 사용할 수 있습니다. 여기에 제시된 프로토콜은 Thordarson 외하여 설명에 따라 달라집니다. percoll 기울기는 절연 마우스 태반에서 비교적 순수한 trophoblast 세포 인구를 얻기 위해 사용되는 1. 그것은 더 널리 사용하는 유사합니다인간 trophoblast 세포 절연 2-3에 D 방법. 순도는 cytokeratin 7 4에 대한 격리 세포의 immunocytochemical 착색에 의해 평가 될 수 있습니다. 여기서, 절연 셀은 다음 체외 5-6에 trophoblast의 invasiveness을 평가하기 위해 matrigel 침공 분석을 사용하여 분석하고 있습니다. 침입 세포는 계산에 대한 immunocytochemistry과 스테인드에 의해 분석됩니다.

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Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

이 프로토콜에서는, 우리는 Percoll 기울기를 사용하여 trophoblast 세포의 분리에 이어 임신 d10.5에 마우스에서 placentae의 절개를 설명합니다. 우리는 matrigel의 침공 분석에서 분리 된 세포의 사용을 보여줍니다.

기본 마우스 Trophoblast 셀 및 Trophoblast의 침략 분석의 절연

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

isolation-primary-mouse-trophoblast-cells-trophoblast-invasion

Research

JoVE Journal

Biology

23.4K 조회수.  University of Missouri. 이 프로토콜에서는, 우리는 Percoll 기울기를 사용하여 trophoblast 세포의 분리에 이어 임신 d10.5에 마우스에서 placentae의 절개를 설명합니다. 우리는 matrigel의 침공 분석에서 분리 된 세포의 사용을 보여줍니다.

비디오: Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Blastocysts에서 마우스 Trophoblast의 줄기 세포의 유도

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

쥐 간장 세포의 분리 및 차 문화

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

일차 종양에서 면역 세포의 분리

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

마우스 기본 췌장 포상 세포의 분리 및 문화

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

직접 레이블이 프로브를 사용하여 강력한 3D DNA 물고기

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

마우스와 인간의 대장 조직에서 주요 근육 섬유 모세포의 분리

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

분리 및 마우스 대동맥 내피 세포의 차 문화

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

마우스 기본 신장 관 상피 세포의 고립, 특성, 및 높은 처리량 세포 외 유출 분석

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

소형 돼지에서 1 차 대동맥 내피 세포의 격리 및 배양

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...